Hepatitis C virus (HCV) causes acute and chronic hepatitis and hepatocellular carcinoma (HCC). Notably, HCV has played a major role in the rise of HCC, accounting for 50% of cases in the United States. Despite an obvious need however, no vaccine is available to protect against HCV infection and only a subset of chronically infected patients respond to current treatment options. As such, there is an obvious and immediate need for new effective HCV antivirals. In particular, HCV entry represents a promising multi-faceted opportunity for drug discovery;however a deeper understanding and coherent biochemical description of the process is still needed to facilitate such endeavors. Importantly, with the recent development of the first robust cell culture HCV infection system, the ability to investigate the molecular details of HCV entry is now possible. As such, the objective of this R03 proposal is to develop live cell imaging research technologies and methodologies for the study of HCV entry by adapting fluorescent labeling strategies that have been successfully utilized in other viral systems for use with the new HCV cell culture infection system. Accordingly, the specific aims of this proposal are: 1) To develop a fluorescence dequenching real-time assay for elucidating the dynamics of HCV-cell fusion in vitro and 2) To develop a fluorescence-based single- virus tracking real-time assay for monitoring the early events of HCV entry and trafficking in vitro. These two different but complimentary approaches will further our understanding of HCV entry dynamics and pave the way for the development of new HCV inhibitors. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects more than 180 million people worldwide, causing acute and chronic hepatitis and hepatocellular carcinoma, however no protective vaccine is available and only a subset of infected patients respond to current treatment options. To design more effective antivirals it is crucial to study the HCV lifecycle to understand the dynamics of infection and identify which steps of infection represent effective antiviral targets. Because HCV entry represents a promising multi-faceted opportunity for drug discovery, we propose to develop and use fluorescence-based real-time assays to investigate the kinetics and conditions that regulate viral entry.